Antibody-dependent Cellular Cytotoxicity Mechanism Research Articles

NK Cell Degranulation Triggered by Rituximab Identifies Potential Markers of Subpopulations with Enhanced Cytotoxicity toward Malignant B Cells.

A promising strategy in cancer immunotherapy is to restore or enhance the cytotoxicity of NK cells, among others, by activating the mechanism of antibody-dependent cellular cytotoxicity (ADCC). Monoclonal antibodies targeting tumor antigens, such as rituximab (targeting CD20), induce NK cell-mediated ADCC and have been used to treat B cell malignancies, such as non-Hodgkin lymphoma, but not always successfully. The aim of this study was to analyze the gene expression profile of the NK cells involved in the cytolytic response stimulated by rituximab. NK cells were co-cultured with rituximab-opsonized Raji cells. Sorting into responder and non-responder groups was based on the presence of CD107a, which is a degranulation marker. RNA-seq results showed that the KIT and TNFSF4 genes were strongly down-regulated in the degranulating population of NK cells (responders); this was further confirmed by qRT-PCR. Both genes encode surface proteins with cellular signaling abilities, namely c-KIT and the OX40 ligand. Consistent with our findings, c-KIT was previously reported to correlate inversely with cytokine production by activated NK cells. The significance of these findings for cancer immunotherapy seems essential, as the pharmacological inhibition of c-KIT and OX40L, or gene ablation, could be further tested for the enhancement of the anti-tumor activity of NK cells in response to rituximab.

Chimeric single-chain variable fragment-human immunoglobulin G crystallizable fragment antibody against GD2 for neuroblastoma targeted immunotherapy

Aim: The present study aims to generate chimeric mouse single-chain variable fragment (scFv) and immunoglobulin G1 (IgG1) crystallizable fragment (Fc) antibody against disialoganglioside (GD2) for the treatment of neuroblastoma (NB). The generated scFv-IgG Fc antibody, lacking first constant domain of heavy chain (CH1), is of a smaller size than the natural antibody and has anti-tumor activity. Methods: Vector for scFv-IgG Fc antibody was constructed and scFv-IgG Fc antibody was expressed in human embryonic kidney 293T (HEK293T) cell line. Purification of scFv-IgG Fc antibody from the culture supernatant of transfected HEK293T cells was performed by Protein G affinity chromatography. The structure and binding activity of scFv-IgG Fc antibody were verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting (WB), and immunofluorescence techniques. Anti-tumor activities by antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) were determined. Results: Using plasmid fusion-human IgG1-Fc2 tag vector (pFUSE-hIgG1-Fc2), a plasmid vector encoding chimeric mouse scFv and hIgG1 Fc antibody against GD2 was successfully constructed. This vector was transfected into human HEK293T cells to produce scFv-IgG Fc antibody. The transfected HEK293T cells could produce chimeric scFv-IgG Fc antibody against GD2, which lacks the IgG heavy chain CH1 domain but carries CH2 and CH3 domains. The chimeric antibodies could be purified from the culture supernatant of the transfected HEK293T culture in the presence of zeocin drug. The produced GD2 scFv-IgG Fc antibodies, which are smaller in size than the intact antibody, could trigger the killing of GD2 expressed NB cell line SH-SY5Y by ADCC and ADCP mechanisms. Conclusions: The results indicate that chimeric scFv-hIgG Fc antibody, lacking heavy chain CH1 domain, could mediate antibody induced anti-tumor activities. The small size of this type of chimeric antibody may be employed as anti-GD2 antibody for NB therapy.

AB-101, an allogeneic, non–genetically modified, natural killer (NK) cell therapy, evaluated as monotherapy or in combination with rituximab in R/R non-Hodgkin lymphoma.

7529 Background: AB-101 is a non-genetically modified, cord blood-derived, allogeneic, cryopreserved, NK cell therapy for use in combination with monoclonal antibody (mAb) or innate cell engager therapies. AB-101 is being developed to enhance response to mAb therapy through the antibody-dependent cellular cytotoxicity (ADCC) mechanism. Many cancer patients (pts) are unable to mount a robust ADCC response, rendering mAb therapy less effective for these pts. AB-101 has attributes that enhance NK cell activity: Consistent phenotype for tumor engagement and cytotoxicity; Selected killer immunoglobulin-like receptor B (KIR B) haplotype; V/V CD16 polymorphism at F158 for enhanced ADCC. AB-101 outpatient administration provides optimized NK cells to pts, potentially enhancing their anti-tumor activity in combination with mAb that utilize the ADCC mechanism. We have initiated a Ph 1/2 clinical trial evaluating the combination of AB-101 with rituximab (RTX) in pts with R/R NHL of B-cell origin. Methods: Two AB-101 dose levels, 1 or 4e9 cells, will be evaluated as monotherapy or combination with RTX. All enrolled pts will receive at least 1 cycle of 4 weekly AB-101 doses, with cyclophosphamide/fludarabine lymphodepletion (LD). Pts receiving the combination may receive up to 4 treatment cycles. Results: Dose escalation is ongoing with 17 pts enrolled. Pts were elderly (median of 68 yrs) and heavily pretreated (median of 4 prior lines) with 64% having had prior CD19 CAR-T. Pts had DLBCL (9), FL (5), MCL (2), and LPL/WM (1). 11 monotherapy pts and 6 RTX combination pts were treated at time of abstract. 16 pts received AB-101 at 1e9 cells/dose; 1 pt received 4e9 cells/dose. 4 of 6 combination pts have received > 1 cycle. AB-101 was generally well tolerated. The most common G3+ AEs were LD-related neutropenia and lymphopenia. All AB-101 related AEs were G1/2. ICANs and GVHD, events associated with allogeneic cell therapy, were not reported; Gr 1 CRS occurred in 2 pts and resolved within 24h. SAEs were reported in 9 pts. The most common SAEs were associated with either infections (n = 3) or PD (n = 3) and one related SAE of Gr 1 fever was reported. No pts discontinued treatment due to an AB-101-related AE. ORR for the RTX cohort was 67% (4/6) with a CR rate of 50% (3/6). 3 of 4 responses were observed in pts who failed prior CAR-T therapy. ORR for the monotherapy cohorts was 27% (3/11). Conclusions: 4 weekly doses of AB-101 given alone or with RTX appeared to be safe and well tolerated without the serious/severe toxicities associated with CAR-T therapies. Preliminary efficacy of the combination treatment is encouraging. The study is ongoing and continues to enroll the combination cohorts and the 4e9 cells/dose level. Updated safety, efficacy, and exploratory PK/PD/immunogenicity results will be provided during the presentation. Clinical trial information: NCT04673617 .

CD46 protects the bladder cancer cells from cetuximab-mediated cytotoxicity

Epidermal growth factor receptor (EGFR) is an effective target for those patients with metastatic colorectal cancers that retain the wild-type RAS gene. However, its efficacy in many cancers, including bladder cancer, is unclear. Here, we studied the in vitro effects of cetuximab monoclonal antibodies (mAbs) targeting EGFR on the bladder cancer cells and role of CD46. Cetuximab was found to inhibit the growth of both colon and bladder cancer cell lines. Furthermore, cetuximab treatment inhibited AKT and ERK phosphorylation in the bladder cancer cells and reduced the expression of CD46 membrane-bound proteins. Restoration of CD46 expression protected the bladder cancer cells from cetuximab-mediated inhibition of AKT and ERK phosphorylation. We hypothesized that CD46 provides protection to the bladder cancer cells against mAb therapies. Bladder cancer cells were also susceptible to cetuximab-mediated immunologic anti-tumor effects. Further, cetuximab enhanced the cell killing by activating both antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in bladder cancer cells. Restoration of CD46 expression protected the cells from both CDC and ADCC induced by cetuximab. Together, CD46 exhibited a cancer-protective effect against both direct (by involvement of PBMC or complement) and indirect cytotoxic activity by cetuximab in bladder cancer cells. Considering its clinical importance, CD46 could be an important link in the action mechanism of ADCC and CDC intercommunication and may be used for the development of novel therapeutic strategies.

Pre-Clinical Efficacy of AB-101, an Allogeneic Cord-Blood Derived Natural Killer (NK) Cell Therapeutic Candidate, in Combination with Anti-CD38 Antibodies in Models of Multiple Myeloma

Introduction: AB-101 is a non-engineered, allogeneic, off-the-shelf, cryopreserved cord blood-derived natural killer (NK)-cell therapy in development as a cancer therapeutic. A highly scaled manufacturing process enables production of 1000s of doses of AB-101 from a single donor cord blood unit (CBU). AB-101 is derived from CBUs selected for KIR-B haplotype and natural high-affinity variant of CD16 (158V/V), which are associated with improved anti-tumor activity and ADCC enhancement, resulting in a highly active NK cell product without the requirement for additional engineering. We are developing AB-101 for combination with monoclonal antibodies (mAbs) to enhance antibody dependent cellular cytotoxicity (ADCC) in patients, initially in an ongoing Phase I clinical trial for NHL. To further develop AB-101 as an ADCC enhancer, we assessed anti-tumor activity in combination with CD38 mAb, against multiple myeloma (MM) cell lines. CD38 is a transmembrane glycoprotein that is uniformly and highly upregulated on MM cells. Anti-CD38 mAbs exert anti-tumor activity against MM through ADCC and other mechanisms. NK cells play a key role in ADCC responses through CD16 receptor signaling. However, the majority of NK cells express CD38 and are depleted by therapeutic CD38 mAbs which could limit their therapeutic efficacy. Combining NK cell therapies with anti-CD38 mAbs, thus necessitates CD38 knockout to prevent fratricide. AB-101 has a naturally low percent and intensity of CD38 expression. Here we present data demonstrating that AB-101 are resistant to anti-CD38 mAb-induced fratricide and can elicit significant ADCC against MM cell lines both in vitro and in vivo. Since anti-CD38 mAbs are combined with glucocorticoids in the clinic, we assessed the impact of dexamethasone and methylprednisolone on ADCC by AB-101. Here we report robust ADCC following treatment of AB-101 with glucocorticoids. Methods: AB-101 was screened for CD38 expression and incubated with anti-CD38 mAb - daratumumab or hIgG1 control (300 - 0.41 µg/ml) for 4 hours to assess fratricide. In vitro ADCC assays were performed at various effector to target ratios against control (Daudi and K562) and MM cell lines (NCI-H929, RPMI 8226, MM.1S and MM.1R) using 2 µg/ml of daratumumab or hIgG1. To study the effect of glucocorticoids, AB-101 was cultured in various concentrations of dexamethasone and methylprednisolone for 48 hours in the presence of 50 or 500 IU/ml of IL-2, prior to ADCC assay. In vivo efficacy of AB-101 in combination with daratumumab was assessed in the intraperitoneal (IP) MM.1S-Luc xenograft model in NSG mice (Figure 1A). Daratumumab and AB-101 were administered by IP injection. Tumor burden was assessed by bioluminescent imaging (BLI). Results: AB-101 was over 98% CD56+/CD3- with 87% CD56+/CD16+ population. Only 22% of GMP drug product were CD38+ with low intensity of expression compared to peripheral blood NK cells (~88%). In the presence of daratumumab AB-101 viability was unaltered compared to hIgG1 control demonstrating resistance to fratricide. Daratumumab enhanced ADCC potential of AB-101 against MM cell lines and Daudi in in vitro. ADCC against MM lines and Daudi did not decrease on exposure to glucocorticoids compared to no treatment group. In vivo anti-tumor efficacy, as evidenced by reduction in BLI signal, was observed in the MM.1S model in AB-101 + daratumumab groups compared to daratumumab and AB-101 alone (Figure 1B). AB-101 + daratumumab (1 and 3mpk) led to 98 and 99% tumor growth inhibition (TGI), respectively, relative to no treatment. Daratumumab monotherapy at 1 and 3mpk showed 21% and 87% TGI respectively, and AB-101 showed 16% TGI relative to no treatment. Conclusions: AB-101 has high and consistent expression of CD16 and low CD38 expression and is resistant to daratumumab induced fratricide and hence can be combined with daratumumab without additional engineering such as CD38 knockout. Daratumumab is combined in the clinic with glucocorticoids, immunomodulatory drugs (IMiDs) and proteosome inhibitors (PI). While IMiDs and PIs are reported to enhance NK cell function, glucocorticoids have been shown to diminish NK cell cytotoxicity. Data presented here suggests that ADCC potential of AB-101 is not negatively impacted by glucocorticoids. Taken together, the in vitro and in vivo data supports further clinical development of AB-101 in combination with daratumumab to treat MM patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Trial in Progress: AB-101, an Allogeneic, Non-Engineered, Cord Blood-Derived Natural Killer (NK) Cell Therapy, As Monotherapy or in Combination with Rituximab in Patients with Relapsed or Refractory Non-Hodgkin Lymphoma

Background: Natural Killer (NK) cells are known to eliminate tumors directly or via antibody-dependent cellular cytotoxicity (ADCC) which is dependent on the maturation, activation and FcγRIIIA (CD16) phenotype of NK cells. Many cancer patients have low NK cell number and dysfunctional innate immunity, which has been correlated with outcomes to monoclonal antibody (mAb) therapy. AB-101 is an allogeneic, non-genetically modified cord blood-derived NK cell therapy, developed to potentially enhance patients' innate anti-tumor activity in combination with approved oncology products with known ADCC mechanism of action. AB-101 has been optimized for combination with mAbs through the novel AlloNK™ proprietary large scale manufacturing process utilizing donor cord blood units. The cord blood unit is selected for key attributes, including an immunoglobulin-like receptor B (KIR-B) haplotype and the natural high-affinity variant of CD16 (158V/V polymorphism). The AlloNK™ process results in an activated and mature NK cell product with increased cell surface expression of CD16, NKG2D, NKp30/44/46, and DNAM-1, without any requirement for genetic engineering. Additionally, the process generates sufficient NK cells to treat hundreds to thousands of patients from a single cord blood unit and produces a consistently active NK cell product, with little donor-to-donor variability. The combination of AB-101 and rituximab has demonstrated strong cytotoxic activity in preclinical studies; the combination led to improved survival over rituximab or NK cells alone in xenograft mouse models. As a cryopreserved, "off-the-shelf", and infusion-ready product, AB-101 is being evaluated for the treatment of hematologic malignancies in combination with standard of care antibody therapies. Study Design, Treatment and Methods: AB-101-01 is a first in human, Phase 1/2, multi-center, open-label study to evaluate the safety and anti-tumor activity of AB-101 as monotherapy and in combination with rituximab in patients with relapsed or refractory (R/R) non-Hodgkin lymphoma (NHL) of B-cell origin or CLL. This study has 3 parts: Phase 1, 1b, and 2. Phase 1 will evaluate 2 dose levels as monotherapy or in combination with rituximab using the 3+3 dose escalation method. Phase 1b will enroll up to 45 patients in combination with rituximab to determine the recommended phase 2 dose (RP2D). Phase 2 will enroll an additional 15 patients at the RP2D. AB-101 will be given at 1 or 4 billion cells per dose. All enrolled patients will receive at least 1 treatment cycle of 4 doses of AB-101, administered once weekly. Prior to receiving the first dose of AB-101, patients will receive 3 consecutive days of lymphodepleting chemotherapy consisting of fludarabine (30 mg/m2/day) and cyclophosphamide (250 or 500 mg/m2/day). After each AB-101 infusion, patients will receive interleukin-2 (IL-2) at 1×106 IU/m2/dose or 6×106 IU/dose to potentiate NK cell activation and persistence. If given, 375 mg/m2 of rituximab is dosed on the second day of lymphodepletion and with the second and fourth dose of AB-101. Patients who have a complete or partial response or maintain stable disease with the first cycle of the combination may receive 3 additional cycles such that cycle 2 is given with lymphodepletion and cycles 3 and 4 do not include lymphodepletion. Eligible patients must have a diagnosis of B-cell NHL or CLL and must have received at least 2 prior lines of therapy, including an anti-CD20 antibody therapy. Treatment with prior CD19 CAR T-cell therapy is permitted. Patients must be at least 18 years of age, have ECOG PS of 0 or 1, and have measurable disease per Lugano 2014 at the time of enrollment. The primary objectives of each phase are as follows: in Phase 1, the evaluation of the safety of 1 and 4 billion cells/dose of AB-101 as monotherapy or in combination with rituximab through the incidence of AEs and DLTs; in Phase 1b, determination of the RP2D based on safety and antitumor response to AB-101 with rituximab; and in Phase 2, further characterization of the efficacy profile. The study is currently enrolling at 10 U.S. centers. (ClinicalTrials.gov Identifier: NCT04673617)

Abstract 506: A homogenous PBMC ADCC bioassay enables bridging studies with ADCC reporter bioassays in immunotherapy monoclonal antibody development

Abstract Measurement of antibody-dependent cellular cytotoxicity (ADCC) is critical for understanding antibody Fc effector functions during monoclonal antibody development. Classic ADCC assays measure the short-term cytotoxicity of target cells, typically pre-loaded with radioactive or fluorescent dyes, after exposure to antibody bound primary PBMCs or NK cells. These assays are widely used in antibody discovery and characterization during early drug development. However, the reproducibility of these assays is not adequate and they have major issues on biosafety or weak assay sensitivity. Moreover, the use of primary effector cells makes them prone to donor variability, low-throughput and therefore not suitable for implementation in quality-control drug development. We previously developed an ADCC reporter bioassay using engineered ADCC effector cells and demonstrated its specificity and ability to measure ADCC mechanism of action. The assay is prequalified according to ICH guidelines; demonstrates precision, accuracy, linearity, and robustness; and is suitable for product release and stability studies in a quality-controlled environment. In order to enable bridging studies comparing primary PBMC-based ADCC assays and ADCC reporter bioassays, we report the development of an improved PBMC ADCC assay using ADCC-prequalified PBMCs and engineered HiBiT target cells for therapeutic monoclonal antibodies with Fc functions. When target cells expressing HaloTag-HiBiT are incubated with an antibody and PBMCs, the target cells are lysed and release HaloTag-HiBiT, which then bind to LgBiT in the detection reagent to form a functional Nano-luciferase to generate Bioluminescence signal. In addition, it can enable the quantitative measurement of antibody-mediated target cell lysis via specific HaloTag staining using a fluorescent HaloTag ligand by flow cytometry. We demonstrate that the new PBMC ADCC assay is simple, homogenous, highly sensitive, and gives a robust assay window, and is capable of measuring potency of rituximab, cetuximab or trastuzumab. Additionally, it shows antibody potency comparable with the ADCC reporter bioassay in ADCC bridging studies. Citation Format: Pete Stecha, Denise Garvin, Julia Gilden, Jun Wang, Jamison Grailer, Jim Hartnett, Gopal B. Krishnan, Frank Fan, Mei Cong, Zhijie Jey Cheng. A homogenous PBMC ADCC bioassay enables bridging studies with ADCC reporter bioassays in immunotherapy monoclonal antibody development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 506.

Complement-independent bystander injury in AQP4-IgG seropositive neuromyelitis optica produced by antibody-dependent cellular cytotoxicity

Cellular injury in AQP4-IgG seropositive neuromyelitis spectrum disorder (herein called NMO) involves AQP4-IgG binding to astrocytes, resulting in astrocyte injury by complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) mechanisms. The rapid disease progression, severe tissue damage, and abundant leukocyte infiltration seen in some NMO patients suggest a more direct mechanism for demyelination and neurologic deficit than secondary injury from astrocyte loss. Here, we report evidence for an ‘ADCC bystander mechanism’ in NMO involving injury to nearby cells by leukocytes following their activation by AQP4-bound AQP4-IgG on astrocytes. In model cocultures containing AQP4-expressing and null CHO cells, AQP4-IgG and complement killed bystander null cells to ~ 100 μm away from AQP4-expressing cells; AQP4-IgG and NK cells produced bystander killing to ~ 300 μm, with perforin deposition seen on injured null cells. Bystander cytotoxicity was also seen with neutrophil-mediated ADCC and in astrocyte-neuron cocultures. Mechanistic studies, including real-time imaging, suggested that leukocytes activated by an AQP4-dependent ADCC mechanism injure bystander cells by direct targeted exocytosis on neighboring cells and not by diffusion of soluble granule contents. In support of this conclusion, ADCC bystander injury was preferentially reduced by an RGDS peptide that inhibits integrin adhesion. Evidence for ADCC bystander injury to oligodendrocytes and neurons was also found in mice following intracerebral injection of AQP4-IgG and NK cells, which was inhibited by RGDS peptide. These results establish a novel cellular pathogenesis mechanism in AQP4-IgG seropositive NMO and provide evidence that inflammatory mechanisms can cause widespread tissue damage in NMO independently of the secondary effects from astrocyte loss.

Abstract 3265: NKTR-255, a polymer-conjugated IL-15 enhances anti-tumor NK cell responses and synergizes with monoclonal antibodies to provide long-term survival in human lymphoma model

Abstract Background: IL-15 is a cytokine that activates and provides survival benefit to NK cells. Exploiting the therapeutic value of native IL-15 has been challenging due to its unfavorable pharmacokinetic properties and tolerability. NKTR-255 is a polymer-conjugated human IL-15 that retains binding affinity to the alpha subunit of IL-15 receptor and exhibits reduced clearance to thereby provide a sustained pharmacodynamics response. NKTR-255 has potential for providing an enhanced immunotherapeutic effect when combined with monoclonal antibodies that mediate tumor killing by antibody dependent cellular cytotoxicity (ADCC). Here we investigate the pharmacological properties of NKTR-255 on NK cells and the therapeutic effect of NKTR-255 when combined with tumor-directed monoclonal antibodies in a B cell lymphoma model. Methods: KHYG-1 cells (human NK cell line) were used to measure phosphorylated STAT5 (pSTAT5) and cell proliferation. Human PBMCs were stimulated with NKTR-255 and/or daratumumab for in vitro NK cell characterization. In the cytotoxic assay, mice received single IV doses of 0.3 mg/kg of NKTR-255 and splenic NK cells were co-cultured with YAC-1 cells (mouse T lymphoma cell line) to measure cytotoxic function. In the Daudi lymphoma model, 1x107 Daudi cells were inoculated IV on Day 0. NKTR-255 (0.03, 0.1 or 0.3 mg/kg SC) was administered on Days 14, 21 and 28 and antibody treatment was administered on Day 14 (daratumumab 0.5 mg/kg IP) or on Days 14 and 17 (rituximab 40 mg/kg IP). Survival rate was determined by onset of hindlimb paralysis as a surrogate parameter. Results: NKTR-255 dose-dependently induced pSTAT5 and proliferation in KHYG-1 cells (EC50 values for pSTAT5: 0.2 ng/ml, proliferation: 5 ng/ml). NKTR-255 also resulted in enhanced pSTAT5 and NKG2D surface expression on human primary NK cells. In addition NKTR-255 increased NK cell degranulation in co-culture experiments with U266 cells (myeloma cell line) with/without daratumumab (anti-human CD38 antibody), as determined by enhanced CD107a surface expression. In vivo pretreatment with NKTR-255 resulted in sustained cytotoxic function of NK cells in both ex vivo and in vivo studies. Finally, NKTR-255 synergistically provided long-term survival benefit in a dose-dependent manner when administered with daratumumab or rituximab (anti-human CD20 antibody) in the Daudi B cell lymphoma model. Conclusions: NKTR-255 is an immune stimulator of NK cells that provides a dose-dependent effect in the proliferation and activation of NK cells. This property of NKTR-255, when administered with daratumumab or rituximab, translates into enhanced therapeutic efficacies of the antibodies in a B cell lymphoma model. These results indicate that combining NKTR-255 with a tumor-directed antibody having an ADCC mechanism can provide a synergistic effect for treating cancers. Citation Format: Takahiro Miyazaki, Saul Kivimäe, Rhoneil Pena, Peiwen Kuo, Marlene Hennessy, Murali Addepalli, Neha Dixit, Wildaliz Nieves, Sara Sheibani, Mekhala Maiti, Laurie VanderVeen, Joanna Wilczek, Loui Madakamutil, Jonathan Zalevsky. NKTR-255, a polymer-conjugated IL-15 enhances anti-tumor NK cell responses and synergizes with monoclonal antibodies to provide long-term survival in human lymphoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3265.

Ex vivo expanded patient-derived γδ T-cell immunotherapy enhances neuroblastoma tumor regression in a murine model

ABSTRACT An effective therapy regimen for relapsed/refractory high-risk neuroblastoma (NB) includes the anti-GD2 monoclonal antibody, dinutuximab, in combination with temozolomide and irinotecan, supporting a role for chemo-immunotherapy in NB. γδ T cells are an attractive anti-tumor immunotherapy because of their direct cytotoxic activity mediated through cell surface receptors NKG2D and CD16. NKG2D facilitates the innate recognition of stress-induced ligands whereas CD16 recognizes antibody bound to tumors and activates mechanisms of antibody-dependent cellular cytotoxicity (ADCC). This study demonstrates an efficient method for expanding and storing γδ T cells from NB patient-derived apheresis products at clinically relevant amounts. The expanded patient-derived γδ T cells were cytotoxic against the K562 cell line and multiple NB cell lines. Combining γδ T cells with dinutuximab led to a 30% increase in tumor cell lysis compared to γδ T cells alone. Furthermore, low-dose temozolomide in combination with expanded γδ T cells and dinutuximab resulted in increased IFNγ secretion and increased γδ T-cell surface expression of FasL and CD107a. IMR5 NB cell line xenografts established subcutaneously in NSG mice were treated with a regimen of dinutuximab, temozolomide, and γδ T cells. This combination caused targeted killing of NB xenografts in vivo, reducing tumor burden and prolonging survival. These data support the continued preclinical testing of dinutuximab and temozolomide in conjunction with γδ T-cell immunotherapy for patients with recurrent/refractory NB.

Abstract B42: The properties of ex vivo expanded γδ T cells provide for the rational use of combination therapies

Abstract Cell-based immunotherapies have relied mainly on the use of αβ T cells or dendritic cells, and technologies to expand and engineer these cells have resulted in potent antitumor products. Although the use of adaptive immunocompetent cells has advanced more rapidly than innate immune cells, such as natural killer or γδ T cells, these alternative cellular products can provide equivalent antitumor activity. However, the methods to expand clinically relevant innate cell products are not as advanced as those for other immunocompetent cells, such as αβ T cells. We have developed a safe and efficient method for expanding, storing, and genetically engineering γδ T cells, and have focused on the use of these ex vivo expanded cells to treat pediatric neuroblastoma. γδ T cells are an attractive candidate for cell-based products because their antitumor activity is directly cytolytic to tumor cells, in part, due to their receptors NKG2D and CD16. NKG2D facilitates the innate recognition of stress-induced ligands such as MICA/B and UL-16 binding proteins (ULBP), and CD16 enables mechanisms of antibody-dependent cellular cytotoxicity (ADCC). We have developed a serum-free expansion protocol using apheresis products from healthy donors and from neuroblastoma patients. γδ T cells from all starting products can be expanded to greater than 70% of the culture within 12-14 days. These γδ T cells are cytotoxic against K562 cells, which has become our standard for which to compare the various expansion products, and 5 human derived neuroblastoma cell lines. We have tested several freezing media and developed a method using serum-free conditions between days 12-14 of expansion, resulting in equivalent cytotoxicity by cells pre- and post-freezing while maintaining greater than 70% viability post-thaw. We also show that our expansion protocol results in γδ T cells that are greater than 90% positive for surface expression of the receptors NKG2D and CD16. Therefore, we are using these cells in combination treatments that include standard chemotherapy regimens for relapsed neuroblastoma patients, i.e., temozolomide (TMZ), and the monoclonal antibody dinutuximab. We have shown that stress antigens such as MICA/B and ULBP2/5/6 are upregulated in vitro on IMR5 cells for 6 hours after a 1-hour exposure to TMZ. Further, combining patient-expanded γδ T cells that express CD16 with a GD2-specific antibody, dinutuximab, induces 30% increased neuroblastoma cell death compared to γδ T cells alone. To test the in vivo effectiveness of 12-day expanded, frozen, and thawed cells, we injected NSG male and female mice with 5 x 106 IMR5 cells subcutaneously. After a palpable 125-mm3 tumor was established, we began treatment with various combinations of γδ T cells, dinutuximab, and TMZ. These ongoing studies show that γδ T cells alone do not provide an antitumor benefit, but tumor regression is achieved when incorporating γδ T cells into dinutuximab and TMZ treatment regimens. This advantage is only observed at lower TMZ doses, whereas at higher TMZ doses, >60 mg/kg, chemotherapy dominates the antitumor effect. Overall, these studies show that a safer, serum-free expanded γδ T cell product can be produced, which can be stored frozen without affecting the cytotoxic properties. The expression of NKG2D and CD16 on the expanded cells allows for the development of combination therapies using cytotoxic chemotherapy agents and antibody-based treatments. The combination of γδ T cells, TMZ, and dinutuximab can provide a rational advancement for treating pediatric neuroblastoma patients, and we hypothesize that this combination therapy is preferred over high-dose chemotherapy regimens because it can reduce treatment-related toxicities and provide a multifaceted immunotherapeutic approach to neuroblastoma treatment. Citation Format: Jaquelyn T. Zoine, Kathryn S. Sutton, Kristopher A. Knight, Kelly C. Goldsmith, Christopher B. Doering, H. Trent Spencer. The properties of ex vivo expanded γδ T cells provide for the rational use of combination therapies [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B42.

P098 A novel flow cytometric assay to simultaneously detect ATG concentration and cytotoxic effect in renal transplant patient

Aim To develop a new flow cytometry assay for detecting ATG concentration and immunokilling effect to monitor immunotherapy in transplantation. Methods CDC: ASHI standard protocol; Flow-ATG: 50ul patient sera or ATG spiked AB sera with and without complement inactivation were incubated with 0.2X106 PBMC for 15’ at 37 C. After 3 washes, the cells were resuspended in 110 ul stain mix containing PE-anti Rabbit IgG, FITC–CD3, PE-Cy7-CD19, and 7-AAD and continuously incubated at RT for an additional 10’. The cells were washed again and acquired on a Canto II flow cytometer. ATG binding (MCS) and cell death % were calculated on total lymphocyte, T, B, and non-T/B lymphocyte populations. Results ATG exerted a dose dependent effect in cell binding and killing on multiple immune cell populations including T, B lymphocytes, and non-T/B cells. T cells were more sensitive to ATG killing than non T cells (Fig. 1). In the binding assay, ATG at 0.4 ng/ml showed significant bindings on T and B lymphocytes and non-T/B cells. The killing effect of ATG was started at 0.25ug/ml and reached a maximum at 1.25ug/ml (100% death rate) on T and non-T cells. After complement inactivation (56C/30’), cytotoxic effect of ATG on immune cells was only slightly decreased (Fig. 2), which indicated the killing of ATG was mainly resulted from mechanisms of antibody-dependent cellular cytotoxicity (ADCC) and apoptosis under our testing system. Five samples from kidney transplant patients undergoing ATG treatment were tested by both CDC and Flow-ATG. The quantitative dynamical changes of ATG binding and killing were observed by Flow-ATG. Compared to the traditional CDC assay Flow-ATG is much more sensitive and also able to measure the concentration of ATG in the same reaction. Conclusions The newly developed Flow-ATG is able to simultaneously detect ATG binding and killing effect on T, B cells, and non-T/B cells. The assay is simple, sensitive (0.4 ng/ml), quantitative, and can be used for monitoring clinical ATG immunotherapy in bone marrow and solid organ transplantations. Download : Download high-res image (263KB) Download : Download full-size image

Abstract 882: The anti-claudin 6 antibody, IMAB027, induces antibody-dependent cellular and complement-dependent cytotoxicity in claudin 6-expressing cancer cells

Abstract Background Claudin 6 (CLDN6) is a tight junction membrane protein whose expression in normal tissue is confined to embryonic cells, but aberrantly expressed in various human cancer types, including some with a high medical need (eg, ovarian and uterine cancers). This tumor-specific expression in adult organs makes CLDN6 an attractive drug target; as such, IMAB027, an anti-CLDN6 monoclonal antibody (mAb), was developed. This report describes the preclinical characteristics of IMAB027. Methods IMAB027 was generated by hybridoma technology; the discovery process was set up so that mAbs that were good binders as well as inducers of the immune effector mechanisms of antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activity would be detected. ADCC and CDC were assessed in vitro. Apoptosis of CLDN6+ cells was assessed by caspase 3/7, annexin V, and TUNEL assays. Xenografted mouse tumors with human CLDN6+ cells were generated to investigate the in vivo antitumor effects of IMAB027 both as a single agent and in combination with chemotherapeutic agents such as paclitaxel. Results: IMAB027 binds specifically to CLDN6 without cross-reactivity with other closely related family members, such as Claudins 3, 4, and 9. IMAB027 induced target-selective ADCC and CDC in a number of CLDN6+ ovarian and testicular cancer cell lines; median EC50 values for ADCC and CDC were of the order of ng/mL in representative cell lines. Direct induction of apoptosis did not appear to be a contributor to the antitumor effect of IMAB027. In cancer cell lines that heterogeneously expressed CLDN6, but not in those with high or homogeneous CLDN6 levels, pretreatment with chemotherapeutic agents upregulated CLDN6 expression. Increased CLDN6 expression sensitized the cells to IMAB027-induced ADCC, resulting in increased cell lysis. In vivo, treatment with IMAB027 was associated with reduced tumor growth and increased overall survival in different mouse tumor models. These potent antitumor effects were observed in both early and advanced ovarian cancer xenografts. IMAB027, in combination with paclitaxel administered to mice with CLDN6+ xenografts, also prolonged survival compared with paclitaxel alone. Conclusions In these preclinical studies, binding of IMAB027 was CLDN6 specific. IMAB027 as a single agent induced cell death in CLDN6+ cancer cells via ADCC and CDC, thereby exerting in vitro and in vivo antitumor activity. Further, in tumors that have heterogeneous CLDN6 expression, chemotherapy sensitizes cells to IMAB027-induced ADCC, and the combination of IMAB027 and chemotherapeutic agents may enhance the antitumor effect of chemotherapy. Citation Format: Özlem Türeci, Maria Kreuzberg, Korden Walter, Stefan Wöll, Ramona Schmitt, Rita Mitnacht-Kraus, Ikumi Nakajo, Tomohiro Yamada, Ugur Sahin. The anti-claudin 6 antibody, IMAB027, induces antibody-dependent cellular and complement-dependent cytotoxicity in claudin 6-expressing cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 882.

Abstract 2772: Preclinical development of a novel antibody-drug conjugate targeting “cold” tumors

Abstract The promise of immunotherapy for cancer is underscored by the recent efficacy of checkpoint inhibitors, which hinder the ability of tumors to escape attack by the immune system. Patients most likely to benefit from such therapy include those whose tumors are inflamed and who express the PD-L1 checkpoint protein. It is imperative that alternative therapies are developed for patients whose tumors do not exhibit these characteristics. Using our proprietary OGAP® system, we identified a novel membrane cancer target, OX001L. OX001L expression in certain cancers is associated with poor prognosis and reduced survival. IHC studies showed substantial prevalence of OX001L across multiple tumor types, with a majority of the OX001L positive samples scoring negative for PD-L1. In non-small cell lung cancer, OX001L expression was significantly increased in tumors lacking abundant intratumoral PD-1 positive T-cell infiltrate or PD-L1 expression compared to inflamed or PD-L1 positive tumors (p<0.00001). This finding suggests that OX001L may play a role in cancer immune escape. In order to effectively target OX001L positive tumor cells, we generated a human OX001L therapeutic antibody which exhibits target selectivity and cross-reactivity to the cynomolgus monkey OX001L orthologue, as demonstrated by FACS. Using immunofluorescence microscopy, we found that the antibody-antigen complex exhibits efficient internalization from the plasma membrane. Glycoengineering improved the antibody's ability to mediate potent antibody dependent cellular cytotoxicity (ADCC) activity in vitro and in vivo. Furthermore, when conjugated to a DNA alkylating toxin, the OX001L antibody promoted highly potent in vitro cytotoxicity of histotypically distinct cancer cell lines and also effected substantial anti-tumor activity in vivo. Importantly, the OX001L antibody and antibody-drug conjugate (ADC) were well tolerated in cynomolgus monkeys. These data indicate that OX001L ADC is a distinctive therapeutic molecule which could act on OX001L positive tumors via both ADC and ADCC mechanisms of action. The ability of the ADC to also promote recruitment and activation of cytotoxic T lymphocytes in OX001L positive tumors, similar to other cytotoxic payloads, will be further explored. This represents a unique therapeutic opportunity to target “cold”/PD-L1 negative, OX001L positive tumors which are less likely to respond to conventional checkpoint inhibitor therapy. In addition, a combination of the OX001L ADC and checkpoint inhibitors could be used to treat OX001L positive, “hot”/PD-L1 positive tumors. Targeting such tumors with both agents (and through two distinct, non-overlapping mechanisms of action) may yield a higher degree of success and/or a decreased propensity for relapse in patients than would checkpoint inhibitor monotherapy. We plan to test this exciting therapeutic opportunity in humanized in vivo models. Citation Format: Angelo Kaplan, Nickolas Attanasio, To Uyen T Do, Sudha Swaminathan, Arnima Bisht, San Lin Lou, Jason Allen, Robert Boyd, James E. Ackroyd, Gleb Feldman, Christian Rohlff, Rachel L. Dusek. Preclinical development of a novel antibody-drug conjugate targeting “cold” tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2772.

Abstract 3944: Combining tumor glypican-3 expression and CD16 Expression on NK cells from peripheral blood to identify patients responding to codrituzumab/GC33/RO5137382

Abstract Background & Aims: Codrituzumab (GC33 or RO5137382), a monoclonal antibody targeting an oncofetal protein glypican-3 (GPC3) expressed on the cell surface of hepatocellular carcinoma (HCC), induces antibody-dependent cellular cytotoxicity (ADCC) and inhibits tumor growth in preclinical studies. Based on this mechanism of codrituzumab, two biomarkers GPC3 and CD16 of the NK cells, which are the effector cells of ADCC, were investigated to correlate with clinical efficacy of codrituzumab in patients with advanced HCC. Methods: Data from a phase II clinical trial of codrituzumab were used for this analysis. GPC3 expression was determined by immunohistochemistry (IHC) analysis of tumor biopsies, and CD16 expression on NK cells were quantified by a flow cytometry analysis of peripheral blood mononuclear cells. Overall survival of patients with high exposure of codrituzumab or placebo (n = 113) was used to compare different patient subgroups stratified by high or low expression of GPC3 and CD16. Results: In individual-biomarker analyses, longer survival after codrituzumab treatment correlates with either high expression level of GPC3 in tumors (n = 97, HR = 0.39, 95%CI = 0.21 - 0.70, p = 0.00081) or a relatively high level of CD16 expressed on NK cells (n = 80, HR = 0.44, 95%CI = 0.23 - 0.83, p = 0.0056) at baseline. Furthermore, joint analyses of the two biomarkers reveal that both high levels of GPC3 and CD16 are required for patients to benefit from codrituzumab treatment (n = 67, HR = 0.29, 95%CI = 0.13 - 0.62, p = 0.00074), and lack of either one of them leads to a loss of codrituzumab therapeutic effect. Conclusions: The retrospective analysis supports the mechanism of ADCC, in which the combination of high GPC3 expression in tumors and high CD16 expression in NK cells from peripheral blood is associated with prolonged overall survival given treatment of codrituzumab. This result supports the usage of both GPC3 and CD16 as potential biomarkers to select HCC patients for codrituzumab treatment. Citation Format: Gong Chen, Ya-Chi Chen, Bernhard Reis, Anton Belousov, Lori Jukofsky, Christine Rossin, Axel Muehlig, Chao Xu, Laurent Essioux, Toshihiko Ohtomo, Laura Di Laurenzio, Oscar Puig, Ray Lee. Combining tumor glypican-3 expression and CD16 Expression on NK cells from peripheral blood to identify patients responding to codrituzumab/GC33/RO5137382. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3944.

Paradox of Protection: Preferential Recognition of CD4-induced Epitopes by Anti-HIV-1 ADCC Antibodies

Paradox of Protection: Preferential Recognition of CD4-induced Epitopes by Anti-HIV-1 ADCC Antibodies

AFM analysis of the multiple types of molecular interactions involved in rituximab lymphoma therapy on patient tumor cells and NK cells

Rituximab is a monoclonal antibody drug approved for the treatment of patients with lymphomas. Rituximab’s main killing mechanism is antibody-dependent cellular cytotoxicity (ADCC). During ADCC, rituximab’s fragment antigen binding (Fab) region binds to the CD20 antigen on the tumor cell and its fragment crystallizable (Fc) region binds to the Fc receptor (FcR) on the natural killer (NK) cells. In this study, two types of molecular interactions (CD20–rituximab, FcR–rituximab) involved in ADCC were measured simultaneously on cells prepared from biopsy specimens of lymphoma patients by utilizing atomic force microscopy (AFM) with functionalized tips carrying rituximab. NK cells were detected by specific NKp46 fluorescent labeling and tumor cells were detected by specific ROR1 fluorescent labeling. Based on the fluorescence recognition, the binding affinity and distribution of FcRs on NK cells, and CD20 on tumor cells, were quantitatively measured and mapped. The binding affinity and distribution of FcRs (on NK cells) and CD20 (on tumor cells) were associated with rituximab clinical efficacy. The experimental results provide a new approach to simultaneously quantify the multiple types of molecular interactions involved in rituximab ADCC mechanism on patient biopsy cells, which is of potential clinical significance to predict rituximab efficacy for personalized medicine.

Antibody-dependent cellular cytotoxicity and cytokine/chemokine secretion by KHYG-1 cells stably expressing FcγRIIIA

Antibody-dependent cellular cytotoxicity (ADCC) mediated by natural killer (NK) cells is a major mechanism of tumor therapy with antibodies. NK cells not only manifest cytotoxicity but also secrete a variety of cytokines/chemokines that regulate immune responses. Using a retroviral vector, in this study we established a KHYG-1 cell line that stably expresses FcγRIIIA (CD16A). The KHYG-1/FcγRIIIA cells exerted potent antibody concentration-dependent ADCC, whereas parental KHYG-1 cells did not. In contrast, without antibody, the natural killer activity of KHYG-1/FcγRIIIA cells was less potent than that of parental KHYG-1 cells. During the course of ADCC, KHYG-1/FcγRIIIA cells secreted IFN-γ and MIP-1α dependent upon antibody concentration, but parental KHYG-1 cells did not. These results suggest that KHYG-1/FcγRIIIA cells would be useful in studies to elucidate the function of NK cells and the mechanism of ADCC.

Real time assays for quantifying cytotoxicity with single cell resolution.

A new live cell-based assay platform has been developed for the determination of complement dependent cytotoxicity (CDC), antibody dependent cellular cytotoxicity (ADCC), and overall cytotoxicity in human whole blood. In these assays, the targeted tumor cell populations are first labeled with fluorescent Cell Tracker dyes and immobilized using a DNA-based adhesion technique. This allows the facile generation of live cell arrays that are arranged arbitrarily or in ordered rectilinear patterns. Following the addition of antibodies in combination with serum, PBMCs, or whole blood, cell death within the targeted population can be assessed by the addition of propidium iodide (PI) as a viability probe. The array is then analyzed with an automated microscopic imager. The extent of cytotoxicity can be quantified accurately by comparing the number of surviving target cells to the number of dead cells labeled with both Cell Tracker and PI. Excellent batch-to-batch reproducibility has been achieved using this method. In addition to allowing cytotoxicity analysis to be conducted in real time on a single cell basis, this new assay overcomes the need for hazardous radiochemicals. Fluorescently-labeled antibodies can be used to identify individual cells that bear the targeted receptors, but yet resist the CDC and ADCC mechanisms. This new approach also allows the use of whole blood in cytotoxicity assays, providing an assessment of antibody efficacy in a highly relevant biological mixture. Given the rapid development of new antibody-based therapeutic agents, this convenient assay platform is well-poised to streamline the drug discovery process significantly.

The Use of Lentinan for Treating Gastric Cancer

Natural compounds containing fungal β-glucans have been used to improve general health for thousands of years in China and Japan. Lentinan, the backbone of β-(1, 3)-glucan with β-(1, 6) branches, is one of the active ingredients purified from Shiitake mushrooms and has been approved as a biological response modifier for the treatment of gastric cancer in Japan. Despite recent advances in chemotherapeutic agents, unresectable or recurrent gastric cancer remains an incurable disease, with survival rates being far from satisfactory. Recent clinical studies have shown that chemo-immunotherapy using lentinan prolongs the survival of patients with advanced gastric cancer, as compared to chemotherapy alone. In addition, trastuzumab, an antibody against HER2/neu growth factor receptor, has been used for the treatment of gastric cancer in combination with cytotoxic chemotherapeutic agents. Lentinan may exert a synergistic action with anti-cancer monoclonal antibodies to activate complement systems through the mechanism of antibody-dependent cellular cytotoxicity and complement dependent cytotoxicity. Because a better understanding of its biological activities should enable us to use lentinan more efficiently in the treatment of gastric cancer, immunological effects provided by β-glucans, a possible mode of action of lentinan, and its clinical application including future potential uses are discussed in the present review.